Angiogenesis plays a critical role in embryonic development and in several physiologic and pathologic conditions, including wound healing, ovulation, diabetic retinopathy and malignancy. In particular, without the nutrients and oxygen provided via this neovascularization, solid tumors would be unable to grow beyond about 2 mm in diameter.
New capillary growth takes place by a series of sequential steps beginning with the dissolution of the capillary basement membrane. Microvascular endothelial cells stimulate a by angiogenic substances, such as basic fibroblast growth factor (bFGF), in vitro secrete collagenase, plasminogen activator, and stromelysin which degrade the basement membrane and allow endothelial cells to migrate toward the angiogenic stimulus. After migrating, the endothelial cells proliferate, develop sprouts, form capillary-like hollow tubules, and finally link tubules into capillary loops.
Basic FGF is a protein which has a molecular weight of approximately 16 kD, is acid- and temperature-sensitive and has a high isoelectric point. A structurally related protein, acidic FGF (aFGF), has an acidic isoelectric point. FGFs exhibit a mitogenic effect on a wide variety of mesenchymal, endocrine and neural cells. Of particular interest is their stimulatory effect on collateral vascularization and angiogenesis. Such mitogenic effects have stimulated considerable interest in FGFs as potential therapeutic agents for wound healing, nerve regeneration and cartilage repair for example.
Many cells that respond to FGF have been shown to possess specific receptors on the cell surface membranes. The receptor proteins appear to be single chain polypeptides with molecular weights ranging from 110 to 165 kD, depending on cell type. The proteins bind basic FGF with high affinity (Kd=10-80 pM), with receptor numbers ranging from 2000 to 80,000 per cell. Such receptors have been purified from rat brain, using a combination of lectin and ligand affinity chromatography and are associated with tyrosine kinase activity, see Imamura et al., B.B.R.C. 155, 583-590 (1989); Huang and Huang, J. Biol. Chem., 261, 9568-9571 (1986).
On baby hamster kidney cells (BHK), two basic FGF receptors with estimated molecular weights of 110 and 130 kD have been reported in Neufeld et al., J. Biol. Chem., 260, 13860-13868 (1985) and Neufeld et al., J. Biol. Chem., 261, 5631-5637 (1986). Both receptor proteins bind basic FGF and acidic FGF, although it appears that the larger receptor protein binds bFGF preferentially and is sometimes referred to as the "high affinity" bFGF receptor; the smaller receptor has somewhat greater affinity for acidic FGF. Other studies have uncovered additional common FGF receptors in cultured cell lines and embryonic tissues which will bind both bFGF and aFGF, see Olwin et al. J. of Cell. Biochem, 39, 443-454 (1989).
The feasibility of using receptor-specific ligands to transport toxins into cells has recently been demonstrated. The strategy, originally applied in immunotherapy by conjugating toxins to monoclonal antibodies (see Blakey et al., Cancer Research, 48, 7072-7078 (1988)), has recently been pursued by coupling toxins with classic endocrine hormones, such as CRF and TRF, with cytokines such as EGF and TGF.alpha. and with lymphokines such as interleukin-2. U.S. Pat. No. 4,468,382 to Bacha et al. shows cytotoxic conjugates having a disulfide bond with a histidine residue to produce a toxic hybrid protein alleged to be useful in the treatment of certain tumors.
Fibroblast growth factor (FGF) has been coupled with cytotoxins to produce FGF conjugates which are mitotoxic. As detailed in Lappi, et al. B.B.R.C. 160, 917-923 (1989), basic FGF has been coupled to saporin-6 (SAP), a ribosome-inactivating protein (RIP) isolated from the seeds of the plant Saponaria officinalis to produce FGF-SAP, which is shown to be a powerful mitotoxin.
Human melanoma is an example of a cancer that has been steadily rising in incidence and is highly refractory to conventional modes of therapy. In Halaban et al., Oncogene Research, 3, 177-186(1988), it was reported that melanoma cells express bFGF transcripts and suggested the bFGF may act as an autocrine growth factor therefor.
A present need exists for developing improved methods of treating melanomas and other cancerous tumors which currently have a low cure rate.